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Abstract:

This invention relates to an isolated nucleic acid fragment encoding a
diacylglycerol acyltransferase. The invention also relates to the
construction of a chimeric gene encoding all or a portion of the
diacylglycerol acyltransferase, in sense or antisense orientation,
wherein expression of the chimeric gene results in production of altered
levels of the diacylglycerol acyltransferase in a transformed host cell.

Claims:

1. An isolated polynucleotide comprising a nucleotide sequence encoding a
first polypeptide of at least 50 amino acids that has at least 60%
identity based on the Clustal method of alignment when compared to a
polypeptide selected from the group consisting of SEQ ID NOs:4, 6, 8, 10,
14, 20 and 22, or an isolated polynucleotide comprising the complement of
the nucleotide sequence.

2. An isolated polynucleotide comprising a nucleotide sequence encoding a
first polypeptide of at least 50 amino acids that has at least 85%
identity based on the Clustal method of alignment when compared to a
polypeptide selected from the group consisting of SEQ ID NOs:18 and 20.

3. An isolated polynucleotide comprising a nucleotide sequence encoding a
first polypeptide of at least 50 amino acids that has at least 80%
identity based on the Clustal method of alignment when compared to a
polypeptide of SEQ ID NO:2.

4. The isolated polynucleotide of claim 1, wherein the isolated
nucleotide sequence consists of a nucleic acid sequence selected from the
group consisting of SEQ ID NOs:1, 7, 13, 15, and 21 that codes for the
polypeptide selected from the group consisting of SEQ ID NOs:2, 8, 14,
16, and 22.

5. The isolated polynucleotide of claim 1 wherein the isolated
polynucleotide is DNA.

6. The isolated polynucleotide of claim 1 wherein the isolated
polynucleotide is RNA.

10. The isolated host cell of claim 7 wherein the isolated host selected
from the group consisting of yeast, bacteria, plant, and virus.

11. A virus comprising the isolated polynucleotide of claim 1.

12. A polypeptide of at least 50 amino acids that has at least 60%
identity based on the Clustal method of alignment when compared to a
polypeptide selected from the group consisting of a diacylglycerol
acyltransferase polypeptide of SEQ ID NOs:4, 6, 8, 10, 14, 20 and 22.

13. A polypeptide of at least 50 amino acids that has at least 85%
identity based on the Clustal method of alignment when compared to a
polypeptide selected from the group consisting of SEQ ID NOs:18 and 20.

14. A polypeptide of at least 50 amino acids that has at least 80%
identity based on the Clustal method of alignment when compared to a
polypeptide of SEQ ID NO:2.

15. A method of selecting an isolated polynucleotide that affects the
level of expression of a diacylglycerol acyltransferase polypeptide in a
plant cell, the method comprising the steps of: (a) constructing an
isolated polynucleotide comprising a nucleotide sequence of at least one
of 30 contiguous nucleotides derived from a nucleotide sequence selected
from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 11, 13, 15, 17, 19,
21, and the complement of such nucleotide sequences; (b) introducing the
isolated polynucleotide into a plant cell; (c) measuring the level of a
polypeptide in the plant cell containing the polynucleotide; and (d)
comparing the level of polypeptide in the plant cell containing the
isolated polynucleotide with the level of polypeptide in a plant cell
that does not contain the isolated polynucleotide.

16. The method of claim 15 wherein the isolated polynucleotide consists
of a nucleotide sequence selected from the group consisting of SEQ ID
NOs:1, 3, 5, 7, 11, 13, 15, 17, 19 and 21 that codes for the polypeptide
selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14,
16, 18, 20 and 22.

17. A method of selecting an isolated polynucleotide that affects the
level of expression of a diacylglycerol acyltransferase polypeptide in a
plant cell, the method comprising the steps of: (a) constructing an
isolated polynucleotide of claim 1; (b) introducing the isolated
polynucleotide into a plant cell; and (c) measuring the level of
diacylglycerol acyltransferase polypeptide in the plant cell containing
the polynucleotide.

18. A method of obtaining a nucleic acid fragment encoding a
diacylglycerol acyltransferase polypeptide comprising the steps of: (a)
synthesizing an oligonucleotide primer comprising a nucleotide sequence
of at least one of 30 contiguous nucleotides derived from a nucleotide
sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, and the complement of such nucleotide sequences;
and (b) amplifying a nucleic acid sequence using the oligonucleotide
primer.

19. A method of obtaining a nucleic acid fragment encoding the amino acid
sequence encoding a diacylglycerol acyltransferase polypeptide comprising
the steps of: (a) probing a cDNA or genomic library with an isolated
polynucleotide comprising a nucleotide sequence of at least one of 30
contiguous nucleotides derived from a nucleotide sequence selected from
the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,
and the complement of such nucleotide sequences; (b) identifying a DNA
clone that hybridizes with the isolated polynucleotide; (c) isolating the
identified DNA clone; and (d) sequencing the cDNA or genomic fragment
that comprises the isolated DNA clone.

20. A method for evaluating at least one compound for its ability to
inhibit the activity of a diacylglycerol acyltransferase, the method
comprising the steps of: (a) transforming a host cell with a chimeric
gene comprising a nucleic acid fragment encoding a diacylglycerol
acyltransferase, operably linked to suitable regulatory sequences; (b)
growing the transformed host cell under conditions that are suitable for
expression of the chimeric gene wherein expression of the chimeric gene
results in production of the diacylglycerol acyltransferase encoded by
the operably linked nucleic acid fragment in the transformed host cell;
(c) optionally purifying the diacylglycerol acyltransferase expressed by
the transformed host cell; (d) treating the diacylglycerol
acyltransferase with a compound to be tested; and (e) comparing the
activity of the diacylglycerol acyltransferase that has been treated with
a test compound to the activity of an untreated diacylglycerol
acyltransferase, thereby selecting compounds with potential for
inhibitory activity.

23. An isolated polynucleotide of claim 1 comprising the nucleotide
sequence comprising at least one of 30 contiguous nucleotides of a
nucleic sequence selected from the group consisting of SEQ ID NOs:1, 3,
5, 7, 9, 11, 13, 15, 17, 19, 21, and the complement of such sequences.

25. A method for positive selection of a transformed cell comprising: (a)
transforming a plant cell with an expression cassette of claim 24; and
(b) growing the transformed plant cell under conditions allowing
expression of the polynucleotide in an amount sufficient to modify oil
content in the plant cell to provide a positive selection means.

Description:

[0001] This application is a continuation of U.S. application Ser. No.
09/856,018, filed May 16, 2001, which is a National Stage Application of
PCT/US99/28354, filed Dec. 1, 1999, which claims the benefit of U.S.
Provisional Application No. 60/110,602, filed Dec. 2, 1998 and U.S.
Provisional Application No. 60/127,111, filed Mar. 31, 1999.

FIELD OF THE INVENTION

[0002] This invention is in the field of plant molecular biology. More
specifically, this invention pertains to nucleic acid fragments encoding
diacylglycerol acyltransferase in plants and seeds.

BACKGROUND OF THE INVENTION

[0003] In eukaryotic cells triacylglycerols are quantitatively the most
important storage form of energy. Acyl CoA:diacylglycerol acyltransferase
(DGAT, EC 2.3.1.20) uses fatty acyl CoA and diacylglycerol as substrates
to catalyze the only committed step in triacylglycerol synthesis. DGAT
plays a fundamental role in the metabolism of cellular glycerolipids.
Because it is an integral membrane protein, DGAT has yet to be purified
to homogeneity. A mouse cDNA encoding a protein with DGAT activity has
been isolated by using a sequence tag clone sharing regions of similarity
with an acyl Co A cholesterol acyltransferase. This mouse DGAT has been
cloned, sequenced and expressed in insect cells and its activity
characterized (Cases, S. et al. (1998) Proc. Natl. Acad. Sci. USA
95:13018-13023).

[0004] DGAT is important for the generation of seed oils, thus
overexpression of DGAT may be useful for increasing oil content of
oilseeds and suppression of DGAT may result in the diversion of carbon
into other metabolites.

SUMMARY OF THE INVENTION

[0005] The present invention relates to a composition comprising an
isolated polynucleotide or polypeptide of the present invention.

[0006] The present invention relates to an isolated polynucleotide of the
present invention comprising the nucleotide sequence comprising at least
one of 30 contiguous nucleotides of a nucleic acid sequence selected from
the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and
21.

[0007] The present invention relates to an expression cassette comprising
an isolated polynucleotide of the present invention operably linked to a
promoter.

[0008] The present invention relates to an isolated polynucleotide
comprising a nucleotide sequence encoding a first polypeptide of at least
50 amino acids that has at least 60% identity based on the Clustal method
of alignment when compared to a polypeptide selected from the group
consisting of SEQ ID NOs:4, 6, 8, 10, 14, 20 and 22 or an isolated
polynucleotide comprising the complement of the nucleotide sequence.

[0009] The present invention relates to an isolated polynucleotide
comprising a nucleotide sequence encoding a first polypeptide of at least
50 amino acids that has at least 85% identity based on the Clustal method
of alignment when compared to a polypeptide selected from the group
consisting of SEQ ID NOs:18 and 20.

[0010] It is preferred that the isolated polynucleotide of the claimed
invention consists of a nucleic acid sequence selected from the group
consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 that
codes for the polypeptide selected from the group consisting of SEQ ID
NOs:2, 6, 8, 10, 14, 16, and 22. The present invention also relates to an
isolated polynucleotide comprising a nucleotide sequences of at least one
of 40 (preferably at least one of 30) contiguous nucleotides derived from
a nucleotide sequence selected from the group consisting of SEQ ID NOs:1,
3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and the complement of such nucleotide
sequences.

[0011] The present invention relates to a chimeric gene comprising an
isolated polynucleotide of the present invention operably linked to
suitable regulatory sequences.

[0012] The present invention relates to an isolated host cell comprising a
chimeric gene of the present invention or an isolated polynucleotide of
the present invention. The host cell may be eukaryotic, such as a yeast
or a plant cell, or prokaryotic, such as a bacterial cell. The present
invention also relates to a virus, preferably a baculovirus, comprising
an isolated polynucleotide of the present invention or a chimeric gene of
the present invention.

[0013] The present invention relates to a process for producing an
isolated host cell comprising a chimeric gene of the present invention or
an isolated polynucleotide of the present invention, the process
comprising either transforming or transfecting an isolated compatible
host cell with a chimeric gene or isolated polynucleotide of the present
invention.

[0014] The present invention relates to a polypeptide of at least 50 amino
acids that has at least 60% identity based on the Clustal method of
alignment when compared to a polypeptide selected from the group
consisting of a diacylglycerol acyltransferase polypeptide of SEQ ID
NOs:4, 6, 8, 10, 14, 20 and 22.

[0015] The present invention relates to a polypeptide of at least 50 amino
acids that has at least 85% identity based on the Clustal method of
alignment when compared to a polypeptide selected from the group
consisting of SEQ ID NOs:18 and 20.

[0016] The present invention relates to a polypeptide of at least 50 amino
acids that has at least 80% identity based on the Clustal method of
alignment when compared to a polypeptide of SEQ ID NO:2.

[0017] The present invention relates to a method of selecting an isolated
polynucleotide that affects the level of expression of a diacylglycerol
acyltransferase polypeptide in a host cell, preferably a plant cell, the
method comprising the steps of: [0018] constructing an isolated
polynucleotide of the present invention or an isolated chimeric gene of
the present invention; [0019] introducing the isolated polynucleotide or
the isolated chimeric gene into a host cell; [0020] measuring the level a
diacylglycerol acyltransferase polypeptide in the host cell containing
the isolated polynucleotide; and [0021] comparing the level of a
diacylglycerol acyltransferase polypeptide in the host cell containing
the isolated polynucleotide with the level of a diacylglycerol
acyltransferase polypeptide in a host cell that does not contain the
isolated polynucleotide.

[0022] The present invention relates to a method of obtaining a nucleic
acid fragment encoding a substantial portion of a diacylglycerol
acyltransferase polypeptide gene, preferably a plant diacylglycerol
acyltransferase polypeptide gene, comprising the steps of: synthesizing
an oligonucleotide primer comprising a nucleotide sequence of at least
one of 40 (preferably at least one of 30) contiguous nucleotides derived
from a nucleotide sequence selected from the group consisting of SEQ ID
NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and the complement of such
nucleotide sequences; and amplifying a nucleic acid fragment (preferably
a cDNA inserted in a cloning vector) using the oligonucleotide primer.
The amplified nucleic acid fragment preferably will encode a portion of a
diacylglycerol acyltransferase amino acid sequence.

[0023] The present invention also relates to a method of obtaining a
nucleic acid fragment encoding all or a substantial portion of the amino
acid sequence encoding a diacylglycerol acyltransferase polypeptide
comprising the steps of: probing a cDNA or genomic library with an
isolated polynucleotide of the present invention; identifying a DNA clone
that hybridizes with an isolated polynucleotide of the present invention;
isolating the identified DNA clone; and sequencing the cDNA or genomic
fragment that comprises the isolated DNA clone.

[0024] A further embodiment of the instant invention is a method for
evaluating at least one compound for its ability to inhibit the activity
of a diacylglycerol acyltransferase, the method comprising the steps of:
(a) transforming a host cell with a chimeric gene comprising a nucleic
acid fragment encoding a diacylglycerol acyltransferase, operably linked
to suitable regulatory sequences; (b) growing the transformed host cell
under conditions that are suitable for expression of the chimeric gene
wherein expression of the chimeric gene results in production of
diacylglycerol acyltransferase in the transformed host cell; (c)
optionally purifying the diacylglycerol acyltransferase expressed by the
transformed host cell; (d) treating the diacylglycerol acyltransferase
with a compound to be tested; and (e) comparing the activity of the
diacylglycerol acyltransferase that has been treated with a test compound
to the activity of an untreated diacylglycerol acyltransferase, thereby
selecting compounds with potential for inhibitory activity.

BRIEF DESCRIPTION OF THE DRAWING AND SEQUENCE DESCRIPTIONS

[0025] The invention can be more fully understood from the following
detailed description and the accompanying drawing and Sequence Listing
which form a part of this application.

[0026] FIGS. 1A, 1B, and 1C show an alignment of the amino acid sequences
from Mus musculus diacylglycerol acetyltransferase (SEQ ID NO:25), the
instant Arabidopsis thaliana diacylglycerol acetyltransferase (araebcF;
SEQ ID NO:2), the instant corn diacylglycerol acetyltransferase
(cpj1c.pk005.h23; SEQ ID NO:8), the instant rice diacylglycerol
acetyltransferase (r1s24.pk0034.d8:fis; SEQ ID NO:14), the instant
soybean diacylglycerol acetyltransferase (sr1.pk0098.a8; SEQ ID NO:16),
and the instant wheat diacylglycerol acetyltransferase
(wr1.pk0119.b6:fis; SEQ ID NO:22). Amino acids which are identical among
all sequences are indicated with an asterisk (*) above the alignment
while those conserved only among the plant sequences are indicated by a
plus sign (+). Dashes are used by the program to maximize alignment of
the sequences.

[0027] Table 1 lists the polypeptides that are described herein, the
designation of the cDNA clones that comprise the nucleic acid fragments
encoding polypeptides representing all or a substantial portion of these
polypeptides, and the corresponding identifier (SEQ ID NO:) as used in
the attached Sequence Listing. The sequence descriptions and Sequence
Listing attached hereto comply with the rules governing nucleotide and/or
amino acid sequence disclosures in patent applications as set forth in 37
C.F.R. §1.821-1.825.

[0028] The nucleotide sequences having SEQ ID NOs:3, 11, 17, and 19 and
the amino acid sequences having SEQ ID NOs:4, 12, 18, and 20 were
presented in the U.S. Provisional Application No. 60/110,602, filed Dec.
2, 1998. The nucleotide sequences having SEQ ID NOs:1 and 15 as well as
the amino acid sequences having SEQ ID NOs:2 and 16 were added in the
U.S. Provisional Application No. 60/127,111, filed Mar. 3, 1999. The
nucleotide sequence presented in SEQ ID NO:15 encodes an entire soybean
diacylglycerol acyltransferase whose amino acid sequence is presented in
SEQ ID NO:16, the amino acid sequence presented in SEQ ID NO:17 encodes
only a portion of the enzyme. The nucleotide sequence presented in SEQ ID
NO:7 corresponds to the full insert sequence and encodes a protein
identical to that of SEQ ID NO:4. The nucleotide sequences presented in
SEQ ID NOs:11 and 19 correspond to a portion of those presented in SEQ ID
NOs:13 and 21.

[0029] The Sequence Listing contains the one letter code for nucleotide
sequence characters and the three letter codes for amino acids as defined
in conformity with the IUPAC-IUBMB standards described in Nucleic Acids
Res. 13:3021-3030 (1985) and in the Biochemical J. 219 (No. 2):345-373
(1984) which are herein incorporated by reference. The symbols and format
used for nucleotide and amino acid sequence data comply with the rules
set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

[0030] In the context of this disclosure, a number of terms shall be
utilized. As used herein, a "polynucleotide" is a nucleotide sequence
such as a nucleic acid fragment. A polynucleotide may be a polymer of RNA
or DNA that is single- or double-stranded, that optionally contains
synthetic, non-natural or altered nucleotide bases. A polynucleotide in
the form of a polymer of DNA may be comprised of one or more segments of
cDNA, genomic DNA, or synthetic DNA. An isolated polynucleotide of the
present invention may include at least one of 40 contiguous nucleotides,
preferably at least one of 30 contiguous nucleotides, most preferably one
of at least 15 contiguous nucleotides, of the nucleic acid sequence of
the SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and the complement
of such sequences.

[0031] As used herein, "contig" refers to a nucleotide sequence that is
assembled from two or more constituent nucleotide sequences that share
common or overlapping regions of sequence homology. For example, the
nucleotide sequences of two or more nucleic acid fragments can be
compared and aligned in order to identify common or overlapping
sequences. Where common or overlapping sequences exist between two or
more nucleic acid fragments, the sequences (and thus their corresponding
nucleic acid fragments) can be assembled into a single contiguous
nucleotide sequence.

[0032] As used herein, "substantially similar" refers to nucleic acid
fragments wherein changes in one or more nucleotide bases results in
substitution of one or more amino acids, but do not affect the functional
properties of the polypeptide encoded by the nucleotide sequence.
"Substantially similar" also refers to nucleic acid fragments wherein
changes in one or more nucleotide bases does not affect the ability of
the nucleic acid fragment to mediate alteration of gene expression by
gene silencing through for example antisense or co-suppression
technology. "Substantially similar" also refers to modifications of the
nucleic acid fragments of the instant invention such as deletion or
insertion of one or more nucleotides that do not substantially affect the
functional properties of the resulting transcript vis-a-vis the ability
to mediate gene silencing or alteration of the functional properties of
the resulting protein molecule. It is therefore understood that the
invention encompasses more than the specific exemplary nucleotide or
amino acid sequences and includes functional equivalents thereof.

[0033] Substantially similar nucleic acid fragments may be selected by
screening nucleic acid fragments representing subfragments or
modifications of the nucleic acid fragments of the instant invention,
wherein one or more nucleotides are substituted, deleted and/or inserted,
for their ability to affect the level of the polypeptide encoded by the
unmodified nucleic acid fragment in a plant or plant cell. For example, a
substantially similar nucleic acid fragment representing at least one of
30 contiguous nucleotides derived from the instant nucleic acid fragment
can be constructed and introduced into a plant or plant cell. The level
of the polypeptide encoded by the unmodified nucleic acid fragment
present in a plant or plant cell exposed to the substantially similar
nucleic fragment can then be compared to the level of the polypeptide in
a plant or plant cell that is not exposed to the substantially similar
nucleic acid fragment.

[0034] For example, it is well known in the art that antisense suppression
and co-suppression of gene expression may be accomplished using nucleic
acid fragments representing less than the entire coding region of a gene,
and by nucleic acid fragments that do not share 100% sequence identity
with the gene to be suppressed. Moreover, alterations in a nucleic acid
fragment which result in the production of a chemically equivalent amino
acid at a given site, but do not effect the functional properties of the
encoded polypeptide, are well known in the art. Thus, a codon for the
amino acid alanine, a hydrophobic amino acid, may be substituted by a
codon encoding another less hydrophobic residue, such as glycine, or a
more hydrophobic residue, such as valine, leucine, or isoleucine.
Similarly, changes which result in substitution of one negatively charged
residue for another, such as aspartic acid for glutamic acid, or one
positively charged residue for another, such as lysine for arginine, can
also be expected to produce a functionally equivalent product. Nucleotide
changes which result in alteration of the N-terminal and C-terminal
portions of the polypeptide molecule would also not be expected to alter
the activity of the polypeptide. Each of the proposed modifications is
well within the routine skill in the art, as is determination of
retention of biological activity of the encoded products. Consequently,
an isolated polynucleotide comprising a nucleotide sequence of at least
one of 60 (preferably at least one of 40, most preferably at least one of
30) contiguous nucleotides derived from a nucleotide sequence selected
from the group consisting of SEQ ID NOs:1, 5, 7, 9, 13, 15, 21, and the
complement of such nucleotide sequences may be used in methods of
selecting an isolated polynucleotide that affects the expression of a
polypeptide in a plant cell. A method of selecting an isolated
polynucleotide that affects the level of expression of a polypeptide such
as diacylglyercol acyltransferase, in a host cell (eukaryotic, such as
plant or yeast, prokaryotic such as bacterial, or viral) may comprise the
steps of: constructing an isolated polynucleotide of the present
invention or an isolated chimeric gene of the present invention;
introducing the isolated polynucleotide or the isolated chimeric gene
into a host cell; measuring the level a polypeptide in the host cell
containing the isolated polynucleotide; and comparing the level of a
polypeptide in the host cell containing the isolated polynucleotide with
the level of a polypeptide in a host cell that does not contain the
isolated polynucleotide.

[0035] Moreover, substantially similar nucleic acid fragments may also be
characterized by their ability to hybridize. Estimates of such homology
are provided by either DNA-DNA or DNA-RNA hybridization under conditions
of stringency as is well understood by those skilled in the art (Hames
and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,
U.K.). Stringency conditions can be adjusted to screen for moderately
similar fragments, such as homologous sequences from distantly related
organisms, to highly similar fragments, such as genes that duplicate
functional enzymes from closely related organisms. Post-hybridization
washes determine stringency conditions. One set of preferred conditions
uses a series of washes starting with 6×SSC, 0.5% SDS at room
temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at
45° C. for 30 min, and then repeated twice with 0.2×SSC,
0.5% SDS at 50° C. for 30 min. A more preferred set of stringent
conditions uses higher temperatures in which the washes are identical to
those above except for the temperature of the final two 30 min washes in
0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred
set of highly stringent conditions uses two final washes in
0.1×SSC, 0.1% SDS at 65° C.

[0036] Substantially similar nucleic acid fragments of the instant
invention may also be characterized by the percent identity of the amino
acid sequences that they encode to the amino acid sequences disclosed
herein, as determined by algorithms commonly employed by those skilled in
this art. Suitable nucleic acid fragments (isolated polynucleotides of
the present invention) encode polypeptides that are at least 70%
identical, preferably at least 80% identical to the amino acid sequences
reported herein. Preferred nucleic acid fragments encode amino acid
sequences that are at least 85% identical to the amino acid sequences
reported herein. More preferred nucleic acid fragments encode amino acid
sequences that are at least 90% identical to the amino acid sequences
reported herein. Most preferred are nucleic acid fragments that encode
amino acid sequences that are at least 95% identical to the amino acid
sequences reported herein. Suitable nucleic acid fragments not only have
the above homologies but typically encode a polypeptide having at least
50 amino acids, preferably at least 100 amino acids, more preferably at
least 150 amino acids, still more preferably at least 200 amino acids,
and most preferably at least 250 amino acids. Sequence alignments and
percent identity calculations were performed using the Megalign program
of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison,
Wis.). Multiple alignment of the sequences was performed using the
Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153)
with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10).
Default parameters for pairwise alignments using the Clustal method were
KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0037] A "substantial portion" of an amino acid or nucleotide sequence
comprises an amino acid or a nucleotide sequence that is sufficient to
afford putative identification of the protein or gene that the amino acid
or nucleotide sequence comprises. Amino acid and nucleotide sequences can
be evaluated either manually by one skilled in the art, or by using
computer-based sequence comparison and identification tools that employ
algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et
al. (1993) J. Mol. Biol. 215:403-410; see also
www.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or more
contiguous amino acids or thirty or more contiguous nucleotides is
necessary in order to putatively identify a polypeptide or nucleic acid
sequence as homologous to a known protein or gene. Moreover, with respect
to nucleotide sequences, gene-specific oligonucleotide probes comprising
30 or more contiguous nucleotides may be used in sequence-dependent
methods of gene identification (e.g., Southern hybridization) and
isolation (e.g., in situ hybridization of bacterial colonies or
bacteriophage plaques). In addition, short oligonucleotides of 12 or more
nucleotides may be used as amplification primers in PCR in order to
obtain a particular nucleic acid fragment comprising the primers.
Accordingly, a "substantial portion" of a nucleotide sequence comprises a
nucleotide sequence that will afford specific identification and/or
isolation of a nucleic acid fragment comprising the sequence. The instant
specification teaches amino acid and nucleotide sequences encoding
polypeptides that comprise one or more particular plant proteins. The
skilled artisan, having the benefit of the sequences as reported herein,
may now use all or a substantial portion of the disclosed sequences for
purposes known to those skilled in this art. Accordingly, the instant
invention comprises the complete sequences as reported in the
accompanying Sequence Listing, as well as substantial portions of those
sequences as defined above.

[0038] "Codon degeneracy" refers to divergence in the genetic code
permitting variation of the nucleotide sequence without effecting the
amino acid sequence of an encoded polypeptide. Accordingly, the instant
invention relates to any nucleic acid fragment comprising a nucleotide
sequence that encodes all or a substantial portion of the amino acid
sequences set forth herein. The skilled artisan is well aware of the
"codon-bias" exhibited by a specific host cell in usage of nucleotide
codons to specify a given amino acid. Therefore, when synthesizing a
nucleic acid fragment for improved expression in a host cell, it is
desirable to design the nucleic acid fragment such that its frequency of
codon usage approaches the frequency of preferred codon usage of the host
cell.

[0039] "Synthetic nucleic acid fragments" can be assembled from
oligonucleotide building blocks that are chemically synthesized using
procedures known to those skilled in the art. These building blocks are
ligated and annealed to form larger nucleic acid fragments which may then
be enzymatically assembled to construct the entire desired nucleic acid
fragment. "Chemically synthesized", as related to nucleic acid fragment,
means that the component nucleotides were assembled in vitro. Manual
chemical synthesis of nucleic acid fragments may be accomplished using
well established procedures, or automated chemical synthesis can be
performed using one of a number of commercially available machines.
Accordingly, the nucleic acid fragments can be tailored for optimal gene
expression based on optimization of nucleotide sequence to reflect the
codon bias of the host cell. The skilled artisan appreciates the
likelihood of successful gene expression if codon usage is biased towards
those codons favored by the host. Determination of preferred codons can
be based on a survey of genes derived from the host cell where sequence
information is available.

[0040] "Gene" refers to a nucleic acid fragment that expresses a specific
protein, including regulatory sequences preceding (5' non-coding
sequences) and following (3' non-coding sequences) the coding sequence.
"Native gene" refers to a gene as found in nature with its own regulatory
sequences. "Chimeric gene" refers any gene that is not a native gene,
comprising regulatory and coding sequences that are not found together in
nature. Accordingly, a chimeric gene may comprise regulatory sequences
and coding sequences that are derived from different sources, or
regulatory sequences and coding sequences derived from the same source,
but arranged in a manner different than that found in nature. "Endogenous
gene" refers to a native gene in its natural location in the genome of an
organism. A "foreign" gene refers to a gene not normally found in the
host organism, but that is introduced into the host organism by gene
transfer. Foreign genes can comprise native genes inserted into a
non-native organism, or chimeric genes. A "transgene" is a gene that has
been introduced into the genome by a transformation procedure.

[0041] "Coding sequence" refers to a nucleotide sequence that codes for a
specific amino acid sequence. "Regulatory sequences" refer to nucleotide
sequences located upstream (5' non-coding sequences), within, or
downstream (3' non-coding sequences) of a coding sequence, and which
influence the transcription, RNA processing or stability, or translation
of the associated coding sequence. Regulatory sequences may include
promoters, translation leader sequences, introns, and polyadenylation
recognition sequences.

[0042] "Promoter" refers to a nucleotide sequence capable of controlling
the expression of a coding sequence or functional RNA. In general, a
coding sequence is located 3' to a promoter sequence. The promoter
sequence consists of proximal and more distal upstream elements, the
latter elements often referred to as enhancers. Accordingly, an
"enhancer" is a nucleotide sequence which can stimulate promoter activity
and may be an innate element of the promoter or a heterologous element
inserted to enhance the level or tissue-specificity of a promoter.
Promoters may be derived in their entirety from a native gene, or be
composed of different elements derived from different promoters found in
nature, or even comprise synthetic nucleotide segments. It is understood
by those skilled in the art that different promoters may direct the
expression of a gene in different tissues or cell types, or at different
stages of development, or in response to different environmental
conditions. Promoters which cause a nucleic acid fragment to be expressed
in most cell types at most times are commonly referred to as
"constitutive promoters". New promoters of various types useful in plant
cells are constantly being discovered; numerous examples may be found in
the compilation by Okamuro and Goldberg (1989) Biochemistry of Plants
15:1-82. It is further recognized that since in most cases the exact
boundaries of regulatory sequences have not been completely defined,
nucleic acid fragments of different lengths may have identical promoter
activity.

[0043] The "translation leader sequence" refers to a nucleotide sequence
located between the promoter sequence of a gene and the coding sequence.
The translation leader sequence is present in the fully processed mRNA
upstream of the translation start sequence. The translation leader
sequence may affect processing of the primary transcript to mRNA, mRNA
stability or translation efficiency. Examples of translation leader
sequences have been described (Turner and Foster (1995) Mol. Biotechnol.
3:225-236).

[0044] The "3' non-coding sequences" refer to nucleotide sequences located
downstream of a coding sequence and include polyadenylation recognition
sequences and other sequences encoding regulatory signals capable of
affecting mRNA processing or gene expression. The polyadenylation signal
is usually characterized by affecting the addition of polyadenylic acid
tracts to the 3' end of the mRNA precursor. The use of different 3'
non-coding sequences is exemplified by Ingelbrecht et al. (1989) Plant
Cell 1:671-680.

[0045] "RNA transcript" refers to the product resulting from RNA
polymerase-catalyzed transcription of a DNA sequence. When the RNA
transcript is a perfect complementary copy of the DNA sequence, it is
referred to as the primary transcript or it may be a RNA sequence derived
from posttranscriptional processing of the primary transcript and is
referred to as the mature RNA. "Messenger RNA (mRNA)" refers to the RNA
that is without introns and that can be translated into polypeptide by
the cell. "cDNA" refers to a double-stranded DNA that is complementary to
and derived from mRNA. "Sense" RNA refers to an RNA transcript that
includes the mRNA and so can be translated into a polypeptide by the
cell. "Antisense RNA" refers to an RNA transcript that is complementary
to all or part of a target primary transcript or mRNA and that blocks the
expression of a target gene (see U.S. Pat. No. 5,107,065, incorporated
herein by reference). The complementarity of an antisense RNA may be with
any part of the specific nucleotide sequence, i.e., at the 5' non-coding
sequence, 3' non-coding sequence, introns, or the coding sequence.
"Functional RNA" refers to sense RNA, antisense RNA, ribozyme RNA, or
other RNA that may not be translated but yet has an effect on cellular
processes.

[0046] The term "operably linked" refers to the association of two or more
nucleic acid fragments on a single nucleic acid fragment so that the
function of one is affected by the other. For example, a promoter is
operably linked with a coding sequence when it is capable of affecting
the expression of that coding sequence (i.e., that the coding sequence is
under the transcriptional control of the promoter). Coding sequences can
be operably linked to regulatory sequences in sense or antisense
orientation.

[0047] The term "expression", as used herein, refers to the transcription
and stable accumulation of sense (mRNA) or antisense RNA derived from the
nucleic acid fragment of the invention. Expression may also refer to
translation of mRNA into a polypeptide. "Antisense inhibition" refers to
the production of antisense RNA transcripts capable of suppressing the
expression of the target protein. "Overexpression" refers to the
production of a gene product in transgenic organisms that exceeds levels
of production in normal or non-transformed organisms. "Co-suppression"
refers to the production of sense RNA transcripts capable of suppressing
the expression of identical or substantially similar foreign or
endogenous genes (U.S. Pat. No. 5,231,020, incorporated herein by
reference).

[0048] "Altered levels" refers to the production of gene product(s) in
transgenic organisms in amounts or proportions that differ from that of
normal or non-transformed organisms.

[0049] "Mature" protein refers to a post-translationally processed
polypeptide; i.e., one from which any pre- or propeptides present in the
primary translation product have been removed. "Precursor" protein refers
to the primary product of translation of mRNA; i.e., with pre- and
propeptides still present. Pre- and propeptides may be but are not
limited to intracellular localization signals.

[0050] A "chloroplast transit peptide" is an amino acid sequence which is
translated in conjunction with a protein and directs the protein to the
chloroplast or other plastid types present in the cell in which the
protein is made. "Chloroplast transit sequence" refers to a nucleotide
sequence that encodes a chloroplast transit peptide. A "signal peptide"
is an amino acid sequence which is translated in conjunction with a
protein and directs the protein to the secretory system (Chrispeels
(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein
is to be directed to a vacuole, a vacuolar targeting signal (supra) can
further be added, or if to the endoplasmic reticulum, an endoplasmic
reticulum retention signal (supra) may be added. If the protein is to be
directed to the nucleus, any signal peptide present should be removed and
instead a nuclear localization signal included (Raikhel (1992) Plant
Phys. 100:1627-1632).

[0052] Standard recombinant DNA and molecular cloning techniques used
herein are well known in the art and are described more fully in Sambrook
et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor
Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Maniatis").

[0053] Nucleic acid fragments encoding at least a portion of several
diacylglycerol acyltransferases have been isolated and identified by
comparison of random plant cDNA sequences to public databases containing
nucleotide and protein sequences using the BLAST algorithms well known to
those skilled in the art. The nucleic acid fragments of the instant
invention may be used to isolate cDNAs and genes encoding homologous
proteins from the same or other plant species. Isolation of homologous
genes using sequence-dependent protocols is well known in the art.
Examples of sequence-dependent protocols include, but are not limited to,
methods of nucleic acid hybridization, and methods of DNA and RNA
amplification as exemplified by various uses of nucleic acid
amplification technologies (e.g., polymerase chain reaction, ligase chain
reaction).

[0054] For example, genes encoding other diacylglycerol acyltransferases,
either as cDNAs or genomic DNAs, could be isolated directly by using all
or a portion of the instant nucleic acid fragments as DNA hybridization
probes to screen libraries from any desired plant employing methodology
well known to those skilled in the art. Specific oligonucleotide probes
based upon the instant nucleic acid sequences can be designed and
synthesized by methods known in the art (Maniatis). Moreover, the entire
sequences can be used directly to synthesize DNA probes by methods known
to the skilled artisan such as random primer DNA labeling, nick
translation, or end-labeling techniques, or RNA probes using available in
vitro transcription systems. In addition, specific primers can be
designed and used to amplify a part or all of the instant sequences. The
resulting amplification products can be labeled directly during
amplification reactions or labeled after amplification reactions, and
used as probes to isolate full length cDNA or genomic fragments under
conditions of appropriate stringency.

[0055] In addition, two short segments of the instant nucleic acid
fragments may be used in polymerase chain reaction protocols to amplify
longer nucleic acid fragments encoding homologous genes from DNA or RNA.
The polymerase chain reaction may also be performed on a library of
cloned nucleic acid fragments wherein the sequence of one primer is
derived from the instant nucleic acid fragments, and the sequence of the
other primer takes advantage of the presence of the polyadenylic acid
tracts to the 3' end of the mRNA precursor encoding plant genes.
Alternatively, the second primer sequence may be based upon sequences
derived from the cloning vector. For example, the skilled artisan can
follow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci.
USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies of the
region between a single point in the transcript and the 3' or 5' end.
Primers oriented in the 3' and 5' directions can be designed from the
instant sequences. Using commercially available 3' RACE or 5' RACE
systems (BRL), specific 3' or 5' cDNA fragments can be isolated (Ohara et
al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989)
Science 243:217-220). Products generated by the 3' and 5' RACE procedures
can be combined to generate full-length cDNAs (Frohman and Martin (1989)
Techniques 1:165). Consequently, a polynucleotide comprising a nucleotide
sequence of at least one of 60 (preferably one of at least 40, most
preferably one of at least 30) contiguous nucleotides derived from a
nucleotide sequence selected from the group consisting of SEQ ID NOs:1,
3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and the complement of such nucleotide
sequences may be used in such methods to obtain a nucleic acid fragment
encoding a substantial portion of an amino acid sequence of a
polypeptide. The present invention relates to a method of obtaining a
nucleic acid fragment encoding a substantial portion of a polypeptide of
a gene (such as diacylglycerol acyltransferases) preferably a substantial
portion of a plant polypeptide of a gene, comprising the steps of:
synthesizing an oligonucleotide primer comprising a nucleotide sequence
of at least one of 60 (preferably at least one of 40, most preferably at
least one of 30) contiguous nucleotides derived from a nucleotide
sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, and the complement of such nucleotide sequences;
and amplifying a nucleic acid fragment (preferably a cDNA inserted in a
cloning vector) using the oligonucleotide primer. The amplified nucleic
acid fragment preferably will encode a portion of a polypeptide.

[0056] Availability of the instant nucleotide and deduced amino acid
sequences facilitates immunological screening of cDNA expression
libraries. Synthetic peptides representing portions of the instant amino
acid sequences may be synthesized. These peptides can be used to immunize
animals to produce polyclonal or monoclonal antibodies with specificity
for peptides or proteins comprising the amino acid sequences. These
antibodies can be then be used to screen cDNA expression libraries to
isolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.
36:1-34; Maniatis).

[0057] The nucleic acid fragments of the instant invention may be used to
create transgenic plants in which the disclosed polypeptides are present
at higher or lower levels than normal or in cell types or developmental
stages in which they are not normally found. This would have the effect
of altering the oil content in those cells.

[0058] Overexpression of the proteins of the instant invention may be
accomplished by first constructing a chimeric gene in which the coding
region is operably linked to a promoter capable of directing expression
of a gene in the desired tissues at the desired stage of development. For
reasons of convenience, the chimeric gene may comprise promoter sequences
and translation leader sequences derived from the same genes. 3'
Non-coding sequences encoding transcription termination signals may also
be provided. The instant chimeric gene may also comprise one or more
introns in order to facilitate gene expression.

[0059] Plasmid vectors comprising the instant chimeric gene can then be
constructed. The choice of plasmid vector is dependent upon the method
that will be used to transform host plants. The skilled artisan is well
aware of the genetic elements that must be present on the plasmid vector
in order to successfully transform, select and propagate host cells
containing the chimeric gene. The skilled artisan will also recognize
that different independent transformation events will result in different
levels and patterns of expression (Jones et al. (1985) EMBO J.
4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), and
thus that multiple events must be screened in order to obtain lines
displaying the desired expression level and pattern. Such screening may
be accomplished by Southern analysis of DNA, Northern analysis of mRNA
expression, Western analysis of protein expression, or phenotypic
analysis.

[0060] For some applications it may be useful to direct the instant
polypeptide to different cellular compartments, or to facilitate its
secretion from the cell. It is thus envisioned that the chimeric gene
described above may be further supplemented by altering the coding
sequence to encode the instant polypeptide with appropriate intracellular
targeting sequences such as transit sequences (Keegstra (1989) Cell
56:247-253), signal sequences or sequences encoding endoplasmic reticulum
localization (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol.
42:21-53), or nuclear localization signals (Raikhel (1992) Plant Phys.
100:1627-1632) added and/or with targeting sequences that are already
present removed. While the references cited give examples of each of
these, the list is not exhaustive and more targeting signals of utility
may be discovered in the future.

[0061] It may also be desirable to reduce or eliminate expression of genes
encoding the instant polypeptides in plants for some applications. In
order to accomplish this, a chimeric gene designed for co-suppression of
the instant polypeptide can be constructed by linking a gene or gene
fragment encoding that polypeptide to plant promoter sequences.
Alternatively, a chimeric gene designed to express antisense RNA for all
or part of the instant nucleic acid fragment can be constructed by
linking the gene or gene fragment in reverse orientation to plant
promoter sequences. Either the co-suppression or antisense chimeric genes
could be introduced into plants via transformation wherein expression of
the corresponding endogenous genes are reduced or eliminated.

[0062] Molecular genetic solutions to the generation of plants with
altered gene expression have a decided advantage over more traditional
plant breeding approaches. Changes in plant phenotypes can be produced by
specifically inhibiting expression of one or more genes by antisense
inhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and
5,283,323). An antisense or cosuppression construct would act as a
dominant negative regulator of gene activity. While conventional
mutations can yield negative regulation of gene activity these effects
are most likely recessive. The dominant negative regulation available
with a transgenic approach may be advantageous from a breeding
perspective. In addition, the ability to restrict the expression of
specific phenotype to the reproductive tissues of the plant by the use of
tissue specific promoters may confer agronomic advantages relative to
conventional mutations which may have an effect in all tissues in which a
mutant gene is ordinarily expressed.

[0063] The person skilled in the art will know that special considerations
are associated with the use of antisense or cosuppression technologies in
order to reduce expression of particular genes. For example, the proper
level of expression of sense or antisense genes may require the use of
different chimeric genes utilizing different regulatory elements known to
the skilled artisan. Once transgenic plants are obtained by one of the
methods described above, it will be necessary to screen individual
transgenics for those that most effectively display the desired
phenotype. Accordingly, the skilled artisan will develop methods for
screening large numbers of transformants. The nature of these screens
will generally be chosen on practical grounds, and is not an inherent
part of the invention. For example, one can screen by looking for changes
in gene expression by using antibodies specific for the protein encoded
by the gene being suppressed, or one could establish assays that
specifically measure enzyme activity. A preferred method will be one
which allows large numbers of samples to be processed rapidly, since it
will be expected that a large number of transformants will be negative
for the desired phenotype.

[0064] The instant polypeptide (or portions thereof) may be produced in
heterologous host cells, particularly in the cells of microbial hosts,
and can be used to prepare antibodies to the these proteins by methods
well known to those skilled in the art. The antibodies are useful for
detecting the polypeptide of the instant invention in situ in cells or in
vitro in cell extracts. Preferred heterologous host cells for production
of the instant polypeptide are microbial hosts. Microbial expression
systems and expression vectors containing regulatory sequences that
direct high level expression of foreign proteins are well known to those
skilled in the art. Any of these could be used to construct a chimeric
gene for production of the instant polypeptide. This chimeric gene could
then be introduced into appropriate microorganisms via transformation to
provide high level expression of the encoded diacylglycerol
acyltransferase. An example of a vector for high level expression of the
instant polypeptide in a bacterial host is provided (Example 7).

[0065] Additionally, the instant polypeptide can be used as a target to
facilitate design and/or identification of inhibitors of those enzymes
that may be useful as herbicides. This is desirable because the
diacylglycerol acyltransferase described herein catalyzes the committed
step in triacylglycerol biosynthesis. Accordingly, inhibition of the
activity of the enzyme described herein could lead to inhibition plant
growth. Thus, the instant diacylglycerol acyltransferase could be
appropriate for new herbicide discovery and design.

[0066] All or a substantial portion of the nucleic acid fragments of the
instant invention may also be used as probes for genetically and
physically mapping the genes that they are a part of, and as markers for
traits linked to those genes. Such information may be useful in plant
breeding in order to develop lines with desired phenotypes. For example,
the instant nucleic acid fragments may be used as restriction fragment
length polymorphism (RFLP) markers. Southern blots (Maniatis) of
restriction-digested plant genomic DNA may be probed with the nucleic
acid fragments of the instant invention. The resulting banding patterns
may then be subjected to genetic analyses using computer programs such as
MapMaker (Lander et al. (1987) Genomics 1:174-181) in order to construct
a genetic map. In addition, the nucleic acid fragments of the instant
invention may be used to probe Southern blots containing restriction
endonuclease-treated genomic DNAs of a set of individuals representing
parent and progeny of a defined genetic cross. Segregation of the DNA
polymorphisms is noted and used to calculate the position of the instant
nucleic acid sequence in the genetic map previously obtained using this
population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).

[0067] The production and use of plant gene-derived probes for use in
genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol.
Biol. Reporter 4:37-41. Numerous publications describe genetic mapping of
specific cDNA clones using the methodology outlined above or variations
thereof. For example, F2 intercross populations, backcross populations,
randomly mated populations, near isogenic lines, and other sets of
individuals may be used for mapping. Such methodologies are well known to
those skilled in the art.

[0069] In another embodiment, nucleic acid probes derived from the instant
nucleic acid sequences may be used in direct fluorescence in situ
hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).
Although current methods of FISH mapping favor use of large clones
(several to several hundred KB; see Laan et al. (1995) Genome Res.
5:13-20), improvements in sensitivity may allow performance of FISH
mapping using shorter probes.

[0070] A variety of nucleic acid amplification-based methods of genetic
and physical mapping may be carried out using the instant nucleic acid
sequences. Examples include allele-specific amplification (Kazazian
(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplified
fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),
allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080),
nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res.
18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet.
7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res.
17:6795-6807). For these methods, the sequence of a nucleic acid fragment
is used to design and produce primer pairs for use in the amplification
reaction or in primer extension reactions. The design of such primers is
well known to those skilled in the art. In methods employing PCR-based
genetic mapping, it may be necessary to identify DNA sequence differences
between the parents of the mapping cross in the region corresponding to
the instant nucleic acid sequence. This, however, is generally not
necessary for mapping methods.

[0071] Loss of function mutant phenotypes may be identified for the
instant cDNA clones either by targeted gene disruption protocols or by
identifying specific mutants for these genes contained in a maize
population carrying mutations in all possible genes (Ballinger and Benzer
(1989) Proc. Natl. Acad. Sci. USA 86:9402-9406; Koes et al. (1995) Proc.
Natl. Acad. Sci. USA 92:8149-8153; Bensen et al. (1995) Plant Cell
7:75-84). The latter approach may be accomplished in two ways. First,
short segments of the instant nucleic acid fragments may be used in
polymerase chain reaction protocols in conjunction with a mutation tag
sequence primer on DNAs prepared from a population of plants in which
Mutator transposons or some other mutation-causing DNA element has been
introduced (see Bensen, supra). The amplification of a specific DNA
fragment with these primers indicates the insertion of the mutation tag
element in or near the plant gene encoding the instant polypeptide.
Alternatively, the instant nucleic acid fragment may be used as a
hybridization probe against PCR amplification products generated from the
mutation population using the mutation tag sequence primer in conjunction
with an arbitrary genomic site primer, such as that for a restriction
enzyme site-anchored synthetic adaptor. With either method, a plant
containing a mutation in the endogenous gene encoding the instant
polypeptide can be identified and obtained. This mutant plant can then be
used to determine or confirm the natural function of the instant
polypeptide disclosed herein.

EXAMPLES

[0072] The present invention is further defined in the following Examples,
in which all parts and percentages are by weight and degrees are Celsius,
unless otherwise stated. It should be understood that these Examples,
while indicating preferred embodiments of the invention, are given by way
of illustration only. From the above discussion and these Examples, one
skilled in the art can ascertain the essential characteristics of this
invention, and without departing from the spirit and scope thereof, can
make various changes and modifications of the invention to adapt it to
various usages and conditions.

Example 1

Composition of cDNA Libraries; Isolation and Sequencing of cDNA Clones

[0073] cDNA libraries representing mRNAs from various Arabidopsis, corn,
rice, soybean, and wheat tissues were prepared. The characteristics of
the libraries are described below.

[0074] cDNA libraries may be prepared by any one of many methods
available. For example, the cDNAs may be introduced into plasmid vectors
by first preparing the cDNA libraries in Uni-ZAP® XR vectors according
to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla,
Calif.). The Uni-ZAP® XR libraries are converted into plasmid
libraries according to the protocol provided by Stratagene. Upon
conversion, cDNA inserts will be contained in the plasmid vector
pBluescript. In addition, the cDNAs may be introduced directly into
precut Bluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New
England Biolabs), followed by transfection into DH10B cells according to
the manufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts
are in plasmid vectors, plasmid DNAs are prepared from randomly picked
bacterial colonies containing recombinant pBluescript plasmids, or the
insert cDNA sequences are amplified via polymerase chain reaction using
primers specific for vector sequences flanking the inserted cDNA
sequences. Amplified insert DNAs or plasmid DNAs are sequenced in
dye-primer sequencing reactions to generate partial cDNA sequences
(expressed sequence tags or "ESTs"; see Adams et al., (1991) Science
252:1651-1656). The resulting ESTs are analyzed using a Perkin Elmer
Model 377 fluorescent sequencer.

Example 2

Identification of cDNA Clones

[0075] cDNA clones encoding diacylglycerol acyltransferases were
identified by conducting BLAST (Basic Local Alignment Search Tool;
Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also
www.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequences
contained in the BLAST "nr" database (comprising all non-redundant
GenBank CDS translations, sequences derived from the 3-dimensional
structure Brookhaven Protein Data Bank, the last major release of the
SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA
sequences obtained in Example 1 were analyzed for similarity to all
publicly available DNA sequences contained in the "nr" database using the
BLASTN algorithm provided by the National Center for Biotechnology
Information (NCBI). The DNA sequences were translated in all reading
frames and compared for similarity to all publicly available protein
sequences contained in the "nr" database using the BLASTX algorithm (Gish
and States (1993) Nat. Genet. 3:266-272) provided by the NCBI. For
convenience, the P-value (probability) of observing a match of a cDNA
sequence to a sequence contained in the searched databases merely by
chance as calculated by BLAST are reported herein as "pLog" values, which
represent the negative of the logarithm of the reported P-value.
Accordingly, the greater the pLog value, the greater the likelihood that
the cDNA sequence and the BLAST "hit" represent homologous proteins.

[0076] The BLASTX search using the EST sequences from clones listed in
Table 3 revealed similarity of the proteins encoded by the cDNAs to a
putative Acyl CoA cholesterol acyltransferase related gene product from
Arabidopsis thaliana (NCBI General Identifier No. 3135276), and to
diacylglycerol acyltransferases from Homo sapiens and Mus musculus (NCBI
General Identifier Nos. 3746533, and 3859934, respectively). Animal acyl
CoA cholesterol acyltransferases have recently been shown to be related
to diacylglycerol acyltransferases (Cases et al. (1998) Proc. Natl. Acad.
Sci. USA 95:13018-13023). The sequences included here are therefore more
likely to be diacylglycerol acyltransferases than acyl CoA cholesterol
acyltransferases since cholesterol is only a very minor constituent of
plant sterols. Shown in Table 3 are the BLAST results for individual ESTs
("EST"), or contigs assembled from two or more ESTs ("Contig"):

[0077] The BLASTX search using the EST sequences from clones listed in
Table 4 revealed similarity of the proteins encoded by the cDNAs to
putative diacylglycerol acyltransferases from Arabidopsis thaliana and
Brassica napus (NCBI General Identifier Nos. 5050913 and 5579408,
respectively). Shown in Table 4 are the BLAST results for the sequences
of the entire cDNA inserts comprising the indicated cDNA clones ("FIS"),
contigs assembled from two or more ESTs ("Contig"), or sequences encoding
the entire protein derived from an FIS and PCR ("CGS"):

[0079] The BLASTX search using the EST sequences from clones listed in
Table 5 revealed similarity of the proteins encoded by the cDNAs to a
hypothetical protein from Arabisopsis thaliana and the Mus musculus DGAT
(NCBI General Identifier Nos: 3135275 and 3859934, respectively). The
sequence of the entire cDNA insert in clone src3c.pk013.h18 was
determined, it was found to contain insertions and deletions with respect
to known diacylglycerol acetyltransferases. Clone srl.pk0098.a8 was found
by searching the DuPont EST database for soybean sequences with
similarities to the entire cDNA sequence from clone src3c.pk013.h18.

[0080] Because it was suspected that the Arabidopsis thaliana putative
ACAT sequence encoded only the C-terminal half of a DGAT, an Arabidopsis
thaliana DGAT sequence was obtained by PCR from a public library
described by Kieber et al. (1993) Cell 72:427-441. This library was
prepared from polyA+ RNA isolated from 3 day-old Arabidopsis thaliana
(Columbia) seedling hypocotyls and consisted of 2 to 3 kb size-selected
cDNA inserts cloned into the EcoRI site of lambda-ZAPII (Stratagene).
Prior to use in PCR reactions, the library was converted into plasmid
form by mass-excision following Hay and Short (1992) Strategies 5:16-18)
to yield pBluescript SK(-)-containing cDNA inserts. Primers used for PCR
were:

[0081] The PCR primers were designed based on EST and genomic sequences in
the public domain. An Arabidopsis thaliana EST sequence (GenBank General
Identifier No. 2414087) was used to design the 3' primer (AT-DGAT3; SEQ
ID NO:23). The 5' primer (AtDGx5; SEQ ID NO:24) was based on Arabidopsis
genomic sequence information found in NCBI General Identifier No.
3135250, but could not have been readily predicted as the appropriate 5'
end of the cDNA, based on public sequences. The 5' primer was designed to
be located upstream of a stop codon located in the same reading frame as
the codon for the putative start methionine. The PCR product from this
primer is therefore likely to contain the entire cDNA.

[0082] Shown in Table 5 are the BLAST results for individual ESTs ("EST"),
or sequences encoding the entire protein derived from an FIS and PCR
("CGS"):

[0084] Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASERGENE bioinformatics
computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the
sequences was performed using the Clustal method of alignment (Higgins
and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP
PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise
alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,
WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and
probabilities indicate that the nucleic acid fragments comprising the
instant cDNA clones encode a substantial portion of three corn, one
entire Arabidopsis, one entire rice, and one entire wheat diacylglycerol
acyltransferase. These sequences represent the first Arabidopsis, corn,
rice, soybean, and wheat sequences encoding diacylglycerol
acyltransferase.

Example 5

Expression of Chimeric Genes in Monocot Cells

[0085] A chimeric gene comprising a cDNA encoding the instant polypeptide
in sense orientation with respect to the maize 27 kD zein promoter that
is located 5' to the cDNA fragment, and the 10 kD zein 3' end that is
located 3' to the cDNA fragment, can be constructed. The cDNA fragment of
this gene may be generated by polymerase chain reaction (PCR) of the cDNA
clone using appropriate oligonucleotide primers. Cloning sites (NcoI or
SmaI) can be incorporated into the oligonucleotides to provide proper
orientation of the DNA fragment when inserted into the digested vector
pML103 as described below. Amplification is then performed in a standard
PCR. The amplified DNA is then digested with restriction enzymes NcoI and
SmaI and fractionated on an agarose gel. The appropriate band can be
isolated from the gel and combined with a 4.9 kb NcoI-SmaI fragment of
the plasmid pML103. Plasmid pML103 has been deposited under the terms of
the Budapest Treaty at ATCC (American Type Culture Collection, 10801
University Blvd., Manassas, Va. 20110-2209), and bears accession number
ATCC 97366. The DNA segment from pML103 contains a 1.05 kb SalI-NcoI
promoter fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalI
fragment from the 3' end of the maize 10 kD zein gene in the vector
pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at 15°
C. overnight, essentially as described (Maniatis). The ligated DNA may
then be used to transform E. coli XL1-Blue (Epicurian Coli XL-1 Blue®;
Stratagene). Bacterial transformants can be screened by restriction
enzyme digestion of plasmid DNA and limited nucleotide sequence analysis
using the dideoxy chain termination method (Sequenase® DNA Sequencing
Kit; U.S. Biochemical). The resulting plasmid construct would comprise a
chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD zein
promoter, a cDNA fragment encoding the instant polypeptide, and the 10 kD
zein 3' region.

[0086] The chimeric gene described above can then be introduced into corn
cells by the following procedure Immature corn embryos can be dissected
from developing caryopses derived from crosses of the inbred corn lines
H99 and LH132. The embryos are isolated 10 to 11 days after pollination
when they are 1.0 to 1.5 mm long. The embryos are then placed with the
axis-side facing down and in contact with agarose-solidified N6 medium
(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept in
the dark at 27° C. Friable embryogenic callus consisting of
undifferentiated masses of cells with somatic proembryoids and embryoids
borne on suspensor structures proliferates from the scutellum of these
immature embryos. The embryogenic callus isolated from the primary
explant can be cultured on N6 medium and sub-cultured on this medium
every 2 to 3 weeks.

[0087] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,
Frankfurt, Germany) may be used in transformation experiments in order to
provide for a selectable marker. This plasmid contains the Pat gene (see
European Patent Publication 0 242 236) which encodes phosphinothricin
acetyl transferase (PAT). The enzyme PAT confers resistance to herbicidal
glutamine synthetase inhibitors such as phosphinothricin. The pat gene in
p35S/Ac is under the control of the 35S promoter from Cauliflower Mosaic
Virus (Odell et al. (1985) Nature 313:810-812) and the 3' region of the
nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium
tumefaciens.

[0088] The particle bombardment method (Klein et al. (1987) Nature
327:70-73) may be used to transfer genes to the callus culture cells.
According to this method, gold particles (1 μm in diameter) are coated
with DNA using the following technique. Ten μg of plasmid DNAs are
added to 50 μL of a suspension of gold particles (60 mg per mL).
Calcium chloride (50 μL of a 2.5 M solution) and spermidine free base
(20 μL of a 1.0 M solution) are added to the particles. The suspension
is vortexed during the addition of these solutions. After 10 minutes, the
tubes are briefly centrifuged (5 sec at 15,000 rpm) and the supernatant
removed. The particles are resuspended in 200 μL of absolute ethanol,
centrifuged again and the supernatant removed. The ethanol rinse is
performed again and the particles resuspended in a final volume of 30
μL of ethanol. An aliquot (5 μL) of the DNA-coated gold particles
can be placed in the center of a Kapton® flying disc (Bio-Rad Labs).
The particles are then accelerated into the corn tissue with a
Biolistic® PDS-1000/He (Bio-Rad Instruments, Hercules Calif.), using a
helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying
distance of 1.0 cm.

[0089] For bombardment, the embryogenic tissue is placed on filter paper
over agarose-solidified N6 medium. The tissue is arranged as a thin lawn
and covered a circular area of about 5 cm in diameter. The petri dish
containing the tissue can be placed in the chamber of the PDS-1000/He
approximately 8 cm from the stopping screen. The air in the chamber is
then evacuated to a vacuum of 28 inches of Hg. The macrocarrier is
accelerated with a helium shock wave using a rupture membrane that bursts
when the He pressure in the shock tube reaches 1000 psi.

[0090] Seven days after bombardment the tissue can be transferred to N6
medium that contains gluphosinate (2 mg per liter) and lacks casein or
proline. The tissue continues to grow slowly on this medium. After an
additional 2 weeks the tissue can be transferred to fresh N6 medium
containing gluphosinate. After 6 weeks, areas of about 1 cm in diameter
of actively growing callus can be identified on some of the plates
containing the glufosinate-supplemented medium. These calli may continue
to grow when sub-cultured on the selective medium.

[0091] Plants can be regenerated from the transgenic callus by first
transferring clusters of tissue to N6 medium supplemented with 0.2 mg per
liter of 2,4-D. After two weeks the tissue can be transferred to
regeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 6

Expression of Chimeric Genes in Dicot Cells

[0092] A seed-specific expression cassette composed of the promoter and
transcription terminator from the gene encoding the 0 subunit of the seed
storage protein phaseolin from the bean Phaseolus vulgaris (Doyle et al.
(1986) J. Biol. Chem. 261:9228-9238) can be used for expression of the
instant polypeptide in transformed soybean. The phaseolin cassette
includes about 500 nucleotides upstream (5') from the translation
initiation codon and about 1650 nucleotides downstream (3') from the
translation stop codon of phaseolin. Between the 5' and 3' regions are
the unique restriction endonuclease sites Nco I (which includes the ATG
translation initiation codon), Sma I, Kpn I and Xba I. The entire
cassette is flanked by Hind III sites.

[0093] The cDNA fragment of this gene may be generated by polymerase chain
reaction (PCR) of the cDNA clone using appropriate oligonucleotide
primers. Cloning sites can be incorporated into the oligonucleotides to
provide proper orientation of the DNA fragment when inserted into the
expression vector. Amplification is then performed as described above,
and the isolated fragment is inserted into a pUC18 vector carrying the
seed expression cassette.

[0094] Soybean embryos may then be transformed with the expression vector
comprising sequences encoding the instant polypeptide. To induce somatic
embryos, cotyledons, 3-5 mm in length dissected from surface sterilized,
immature seeds of the soybean cultivar A2872, can be cultured in the
light or dark at 26° C. on an appropriate agar medium for 6-10
weeks. Somatic embryos which produce secondary embryos are then excised
and placed into a suitable liquid medium. After repeated selection for
clusters of somatic embryos which multiplied as early, globular staged
embryos, the suspensions are maintained as described below.

[0095] Soybean embryogenic suspension cultures can maintained in 35 mL
liquid media on a rotary shaker, 150 rpm, at 26° C. with
florescent lights on a 16:8 hour day/night schedule. Cultures are
subcultured every two weeks by inoculating approximately 35 mg of tissue
into 35 mL of liquid medium.

[0096] Soybean embryogenic suspension cultures may then be transformed by
the method of particle gun bombardment (Klein et al. (1987) Nature
(London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic®
PDS1000/HE instrument (helium retrofit) can be used for these
transformations.

[0097] A selectable marker gene which can be used to facilitate soybean
transformation is a chimeric gene composed of the 35S promoter from
Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), the
hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;
Gritz et al. (1983) Gene 25:179-188) and the 3' region of the nopaline
synthase gene from the T-DNA of the Ti plasmid of Agrobacterium
tumefaciens. The seed expression cassette comprising the phaseolin 5'
region, the fragment encoding the instant polypeptide and the phaseolin
3' region can be isolated as a restriction fragment. This fragment can
then be inserted into a unique restriction site of the vector carrying
the marker gene.

[0098] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added
(in order): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and
50 μL CaCl2 (2.5 M). The particle preparation is then agitated
for three minutes, spun in a microfuge for 10 seconds and the supernatant
removed. The DNA-coated particles are then washed once in 400 μL 70%
ethanol and resuspended in 40 μL of anhydrous ethanol. The
DNA/particle suspension can be sonicated three times for one second each.
Five μL of the DNA-coated gold particles are then loaded on each macro
carrier disk.

[0099] Approximately 300-400 mg of a two-week-old suspension culture is
placed in an empty 60×15 mm petri dish and the residual liquid
removed from the tissue with a pipette. For each transformation
experiment, approximately 5-10 plates of tissue are normally bombarded.
Membrane rupture pressure is set at 1100 psi and the chamber is evacuated
to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5
inches away from the retaining screen and bombarded three times.
Following bombardment, the tissue can be divided in half and placed back
into liquid and cultured as described above.

[0100] Five to seven days post bombardment, the liquid media may be
exchanged with fresh media, and eleven to twelve days post bombardment
with fresh media containing 50 mg/mL hygromycin. This selective media can
be refreshed weekly. Seven to eight weeks post bombardment, green,
transformed tissue may be observed growing from untransformed, necrotic
embryogenic clusters. Isolated green tissue is removed and inoculated
into individual flasks to generate new, clonally propagated, transformed
embryogenic suspension cultures. Each new line may be treated as an
independent transformation event. These suspensions can then be
subcultured and maintained as clusters of immature embryos or regenerated
into whole plants by maturation and germination of individual somatic
embryos.

Example 7

Expression of Chimeric Genes in Microbial Cells

[0101] The cDNAs encoding the instant polypeptide can be inserted into the
T7 E. coli expression vector pBT430. This vector is a derivative of
pET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs the
bacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 was
constructed by first destroying the EcoR I and Hind III sites in pET-3a
at their original positions. An oligonucleotide adaptor containing EcoR I
and Hind III sites was inserted at the BamH I site of pET-3a. This
created pET-3aM with additional unique cloning sites for insertion of
genes into the expression vector. Then, the Nde I site at the position of
translation initiation was converted to an Nco I site using
oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM in this
region, 5'-CATATGG, was converted to 5'-CCCATGG in pBT430.

[0102] Plasmid DNA containing a cDNA may be appropriately digested to
release a nucleic acid fragment encoding the protein. This fragment may
then be purified on a 1% NuSieve GTG® low melting agarose gel (FMC).
Buffer and agarose contain 10 μg/ml ethidium bromide for visualization
of the DNA fragment. The fragment can then be purified from the agarose
gel by digestion with GELase® (Epicentre Technologies) according to
the manufacturer's instructions, ethanol precipitated, dried and
resuspended in 20 μL of water. Appropriate oligonucleotide adapters
may be ligated to the fragment using T4 DNA ligase (New England Biolabs,
Beverly, Mass.). The fragment containing the ligated adapters can be
purified from the excess adapters using low melting agarose as described
above. The vector pBT430 is digested, dephosphorylated with alkaline
phosphatase (NEB) and deproteinized with phenol/chloroform as described
above. The prepared vector pBT430 and fragment can then be ligated at
16° C. for 15 hours followed by transformation into DH5
electrocompetent cells (GIBCO BRL). Transformants can be selected on agar
plates containing LB media and 100 μg/mL ampicillin. Transformants
containing the gene encoding the instant polypeptide are then screened
for the correct orientation with respect to the T7 promoter by
restriction enzyme analysis.

[0103] For high level expression, a plasmid clone with the cDNA insert in
the correct orientation relative to the T7 promoter can be transformed
into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol.
189:113-130). Cultures are grown in LB medium containing ampicillin (100
mg/L) at 25° C. At an optical density at 600 nm of approximately
1, IPTG (isopropylthio-β-galactoside, the inducer) can be added to a
final concentration of 0.4 mM and incubation can be continued for 3 h at
25°. Cells are then harvested by centrifugation and re-suspended
in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM
phenyl methylsulfonyl fluoride. A small amount of 1 mm glass beads can be
added and the mixture sonicated 3 times for about 5 seconds each time
with a microprobe sonicator. The mixture is centrifuged and the protein
concentration of the supernatant determined. One μg of protein from
the soluble fraction of the culture can be separated by
SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein
bands migrating at the expected molecular weight.

Example 8

Evaluating Compounds for Their Ability to Inhibit the Activity of
Diacylglycerol Acyltransferases

[0104] The polypeptide described herein may be produced using any number
of methods known to those skilled in the art. Such methods include, but
are not limited to, expression in bacteria as described in Example 7, or
expression in eukaryotic cell culture, in planta, and using viral
expression systems in suitably infected organisms or cell lines. The
instant polypeptide may be expressed either as mature forms of the
proteins as observed in vivo or as fusion proteins by covalent attachment
to a variety of enzymes, proteins or affinity tags. Common fusion protein
partners include glutathione S-transferase ("GST"), thioredoxin ("Trx"),
maltose binding protein, and C- and/or N-terminal hexahistidine
polypeptide ("(His)6"). The fusion proteins may be engineered with a
protease recognition site at the fusion point so that fusion partners can
be separated by protease digestion to yield intact mature enzyme.
Examples of such proteases include thrombin, enterokinase and factor Xa.
However, any protease can be used which specifically cleaves the peptide
connecting the fusion protein and the enzyme.

[0105] Purification of the instant polypeptide, if desired, may utilize
any number of separation technologies familiar to those skilled in the
art of protein purification. Examples of such methods include, but are
not limited to, homogenization, filtration, centrifugation, heat
denaturation, ammonium sulfate precipitation, desalting, pH
precipitation, ion exchange chromatography, hydrophobic interaction
chromatography and affinity chromatography, wherein the affinity ligand
represents a substrate, substrate analog or inhibitor. When the instant
polypeptide are expressed as fusion proteins, the purification protocol
may include the use of an affinity resin which is specific for the fusion
protein tag attached to the expressed enzyme or an affinity resin
containing ligands which are specific for the enzyme. For example, the
instant polypeptide may be expressed as a fusion protein coupled to the
C-terminus of thioredoxin. In addition, a (His)6 peptide may be
engineered into the N-terminus of the fused thioredoxin moiety to afford
additional opportunities for affinity purification. Other suitable
affinity resins could be synthesized by linking the appropriate ligands
to any suitable resin such as Sepharose-4B. In an alternate embodiment, a
thioredoxin fusion protein may be eluted using dithiothreitol; however,
elution may be accomplished using other reagents which interact to
displace the thioredoxin from the resin. These reagents include
β-mercaptoethanol or other reduced thiol. The eluted fusion protein
may be subjected to further purification by traditional means as stated
above, if desired. Proteolytic cleavage of the thioredoxin fusion protein
and the enzyme may be accomplished after the fusion protein is purified
or while the protein is still bound to the ThioBond® affinity resin or
other resin.

[0106] Crude, partially purified or purified enzyme, either alone or as a
fusion protein, may be utilized in assays for the evaluation of compounds
for their ability to inhibit enzymatic activation of the instant
polypeptide disclosed herein. Assays may be conducted under well known
experimental conditions which permit optimal enzymatic activity. For
example, assays for diacylglycerol acyltransferases are presented by M.
Andersson et al. ((1994) J. Lipid Res. 35:535-545).